The long-term goal of this application is to understand the mechanism of action of topoisomerase I (Top1)-targeting drugs. Camptothecins (CPTs) (e.g. irinotecan and topotecan), the prototypic Top1-targeting drugs, have been developed into the clinic. Despite their extraordinary and broad-spectrum antitumor activities observed in mouse models, clinical use of CPTs has so far been limited to only a few tumor types. In order to accelerate the clinical development of Top1-targeting drugs, a detailed understanding of their mechanism of action and various drug resistance mechanisms is urgently needed. Our preliminary studies have suggested that ISG15 (interferon-stimulated gene- 15), a new tumor marker, may be involved in a novel mechanism for CPT sensitivity in tumor cells. ISG15 is a ubiquitin-like protein (UBL) that is induced by type I interferons (IFNs). Similar to ubiquitin, ISG15 is conjugated to many protein substrates (ISGylation) requiring E1, E2 and E3s, all of which are interferon-inducible. ISG15 is highly elevated in many types of tumors, but absent in normal cells (except endometrium during early pregnancy). Significantly, the expression levels of ISG15 have been positively correlated with disease progression in bladder cancer patients. Moreover, ISG15 has been suggested to be a potential prognostic marker for breast cancer. In addition to the significance of ISG15 in tumorigenesis, we have shown that elevated expression of ISG15 in tumor cells confers CPT sensitivity since knocking down either ISG15 or UbcH8 (the major E2 for ISG15) in breast cancer cells results in increased resistance to CPT. The molecular basis for the role of ISG15 in CPT sensitivity is unclear. However, the possible involvement of the ubiquitin-proteasome system is suggested from the following two observations: (1) elevated expression of ISG15 in tumor cells has been shown to antagonize the ubiquitin-proteasome pathway by blocking protein polyubiquitination, and (2) a ubiquitin-proteasome pathway is uniquely involved in the repair of CPT-induced DNA damage. Based on these and other results, we hypothesize that the elevated expression of the ISG15 conjugation pathway in tumor cells inhibits proteasome-mediated repair of CPT-induced DNA damage, resulting in increased sensitivity of tumor cells to CPTs. In the current proposal, we plan to firmly establish that elevated expression of ISG15 and/or the ISG15 conjugation pathway represents a novel mechanism for CPT sensitivity using both cell culture and mouse models, and to elucidate its underlying molecular mechanism. Successful completion of the proposed studies will not only advance our understanding of the mechanism of action of Top1- targeting drugs but also may lead to the development of better treatment strategies for Top1 drug-based cancer therapy. The specific aims are (1) to determine the role of ISG15 and/or ISGylation in CPT sensitivity in both tissue culture and animal models, and (2) to elucidate the molecular mechanism underlying the modulatory role of ISG15 and/or ISGylation in CPT sensitivity. PUBLIC HEALTH RELEVANCE: The long-term goal of this application is to understand the mechanism of action of topoisomerase I (Top1)-targeting drugs. Camptothecins (CPTs) (e.g. irinotecan and topotecan), the prototypic Top1-targeting drugs, have been developed into the clinic. Despite their extraordinary and broad-spectrum antitumor activities observed in mouse models, clinical use of CPTs has so far been limited to only a few tumor types. In order to accelerate the clinical development of Top1-targeting drugs, a detailed understanding of their mechanism of action and drug resistance is urgently needed. Our preliminary studies have suggested that ISG15 (interferon-stimulated gene 15) is a new tumor marker and could be a biomarker for tumor cell sensitivity to Top1 targeting drugs. Our proposed research will firmly establish the role of ISG15 in CPT sensitivity, as well as its underlying mechanism. Successful completion of the proposed studies will not only advance our understanding of the mechanism of action of Top1-targeting drugs but also may lead to development of better treatment strategies for Top1 drug-based cancer therapy.